Cloning vector
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Advances in Cloning Vector Technologies
Introduction to Cloning Vectors
Cloning vectors are essential tools in molecular biology, enabling the insertion, replication, and expression of foreign DNA in host cells. Recent advancements have introduced various innovative cloning strategies and vector systems, enhancing the efficiency and versatility of genetic engineering.
Universal Mini-Vector for Multiple Cloning Strategies
A novel mini-vector, pANY1, has been developed to support multiple cloning strategies, including sticky-end ligation, TA cloning, blunt-end ligation, and ligase-independent cloning. This vector addresses the challenge of selecting appropriate restriction sites by introducing an annealing of PCR products (APP)-based sticky-end cloning strategy. Additionally, pANY1 contains a ccdB cassette between multiple cloning sites to reduce false positives, making it a valuable tool for diverse applications in biosciences and biotechnology1.
USER Cloning for Mammalian Cell Engineering
A versatile mammalian vector system utilizing USER cloning has been designed for rapid construction of expression vectors. This system allows the assembly of up to seven DNA fragments in a single step with high efficiency. It includes a toolbox of DNA building blocks, such as promoters, terminators, and selectable markers, which can be easily combined using Flexible Assembly Sequence Tags (FASTs). This platform facilitates high-throughput studies and customizable gene expression in mammalian cells2.
Ligation-Independent Cloning for High-Throughput Screening
The In-Fusion™ cloning system offers a ligation-independent method suitable for high-throughput expression screening. This system is efficient over a wide range of insert concentrations and supports cloning in multiple hosts, including E. coli and eukaryotic cells. It enables precise engineering of tagged constructs without adding undesirable amino acids, streamlining the process of protein expression and purification3.
Efficient Construction of Lentiviral Vectors
Lentiviral vectors (LVs) are crucial for transgene expression but are often challenging to construct due to their large size. A combinatorial strategy involving site-directed mutagenesis and optimized DNA ratios has been developed to enhance the efficiency of LV construction. This method significantly increases the percentage of positive clones, facilitating the creation of large-size vectors for various applications4.
Transformation-Competent Artificial Chromosome (TAC) Vectors
The pYLTAC7 vector system has been designed for positional cloning in plants, capable of maintaining large genomic DNA fragments in both E. coli and Agrobacterium tumefaciens. This system supports chromosome walking and genetic complementation, enabling precise gene isolation and functional studies in plants. The TAC vector has been successfully used to complement mutants and map genes with high precision5.
Golden Gate Cloning for Plant Synthetic Biology
Golden Gate cloning provides a modular approach for assembling multigene constructs in plants. A comprehensive toolbox of standardized parts, including promoters, tags, and selectable markers, has been developed to facilitate the construction of complex genetic assemblies. This method allows for efficient and precise multigene cloning, enhancing the capabilities of plant synthetic biology6.
Sequence and Ligation Independent Cloning (SLIC)
SLIC cloning offers a fast and efficient method for parallel cloning of expression vectors. This approach uses homologous recombination to join vector and insert DNA with high precision. The inclusion of the toxic ccdB gene for counterselection ensures a background-free cloning process. This system has been successfully applied to various expression vectors, demonstrating high cloning efficiency and versatility7.
Seamless Cloning with Modified pET Vectors
A modification of the pET29 expression vector has been introduced to enable seamless cloning via a gene replacement and Golden Gate strategy. This modification supports the rapid cloning of bacterial enzyme variants from genomic and metagenomic sources, facilitating applications in biocatalysis and other fields8.
Polycistronic Vectors with 2A Peptides
The use of 2A "self-cleaving" peptides in polycistronic vectors allows for the co-expression of multiple genes. Systematic comparisons of different 2A peptides have shown that their position within the construct affects protein expression levels. This strategy has been optimized for applications such as direct cardiac reprogramming, demonstrating the utility of 2A peptides in both basic and translational research9.
Conclusion
The development of advanced cloning vectors and strategies has significantly enhanced the efficiency and flexibility of genetic engineering. From universal mini-vectors to sophisticated systems for mammalian and plant cells, these innovations are driving progress in molecular biology, biotechnology, and synthetic biology.
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Most relevant research papers on this topic
A universal mini-vector and an annealing of PCR products (APP)-based cloning strategy for convenient molecular biological manipulations.
The mini-vector pANY1 enables convenient molecular biological manipulations by meeting the requirements of various cloning strategies and avoiding false positives.
A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering
The versatile mammalian vector system allows rapid construction of customizable expression vectors for protein production, cell biology analyses, and cell factory engineering, facilitating high-throughput genome-scale studies.
Rigorous JournalA versatile ligation-independent cloning method suitable for high-throughput expression screening applications
This versatile ligation-independent cloning method allows for high-throughput expression screening in multiple hosts, enabling precise engineering of (His6-) tagged constructs without undesirable vector or restriction-site-derived amino acids.
Highly CitedQuantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site
This study developed an efficient method for constructing large-size lentiviral vectors with a unique clone site, resulting in a 48%-7.6% positive cloning rate.
In Vitro StudyHighly CitedComplementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning.
The transformation-competent artificial chromosome vector pYLTAC7 enables precise and efficient positional cloning in plants, enabling the precise location of gene mutations.
Highly CitedA golden gate modular cloning toolbox for plants.
The Golden Gate modular cloning toolbox for plants enables efficient assembly of multigene constructs for plant transformation, with comparative performance in Nicotiana benthamiana.
Highly CitedA new method to customize protein expression vectors for fast, efficient and background free parallel cloning
This new expression vector series allows efficient and cost-effective parallel cloning of different protein constructs, tags, and expression hosts, enabling precise engineering of proteins.
In Vitro StudyHighly CitedpET expression vector customized for efficient seamless cloning.
The modified pET29 expression vector allows for rapid and straightforward parallel cloning of bacterial natural enzyme variants for biocatalysis applications.
Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector
Variation in gene position and 2A peptides in polycistronic vectors can improve the efficiency of cloning multiple genes in a single vector, potentially benefiting both basic research and clinical applications.
In Vitro StudyHighly CitedRapid and efficient cosmid cloning
This method allows rapid and efficient cloning of individual DNA fragments, with an average insert size of 38kb, and a rapid and efficient method for preparing plasmid and cosmid DNA.
Highly Cited